Stem cells are slow-cycling, undifferentiated, or immature cells that are capable of giving rise to specialized cell types and ultimately to differentiated cells. These differentiated cells comprise the fully functional organs and tissues within the adult animal and are the end-product of embryonic development. Stem cells have two main characteristics. First, unlike any other cells, they are capable of replenishing tissues by generating, dividing and differentiating. Often, stem cells are multipotent, able to give rise to more than one type of mature cell/tissue. Second, stem cells are also able to renew themselves so that an essentially endless supply of mature cell types can be generated when needed. Because of this capacity for self-renewal, stem cells are therapeutically useful for the regeneration and repair of tissues.
The potency of a stem cell is measured by the variety of different cell types it can ultimately produce. The most potent stem cell is the pluripotent stem cell which can give rise to all cell types of the body (Wagner (1990) EMBO J. 9:3025-3032; Matsui et al. (1992) Cell 70:841-847; Resnick et al. (1992) Nature 359:550-551). Other stem cells exist and include multipotent stem cells which give rise to two or more different cell types. For example, the multipotent hematopoietic stem cell is capable of giving rise to all cell types of the blood system (Jones et al. (1990) Nature 347:188-189; Fleming et al. (1993) J. Cell Biol. 122:897-902). Other known multipotent stem cells include a neuronal stem cell, a neural crest stem cell (Reynolds and Weiss (1992) Science 255:1707-1710; Stemple and Anderson (1992) Cell 71:973-985), and a hair follicle stem cell (Taylor et al. (2000) Cell 102:451). Bipotential stem cells are also considered multipotent stem cells since they give rise to more than one cell type. Specific examples of bipotential stem cells include the O-2A progenitor (Lillien and Raff (1990) Neuron 5:111-119; McKay (1989) Cell 58:815-821; Wolswijk and Noble (1989) Development 105:387-400) and the sympathoadrenal stem cell (Patterson (1990) Cell 62:1035-1038). An example of a monopotent stem cell is the stem cell that resides in the epidermis (Jones and Watt (1993) Cell 73:713-723).
The usefulness of stem cells for tissue regeneration and repair has been shown in several systems. For example, grafting of a hematopoietic stem cell has been shown to rescue an animal which has had its bone marrow subjected to lethal doses of radiation (Jones et al. (1990) supra). An O-2A progenitor has also been shown to remyelinate spinal cord neurons that have been chemically demyelinated (Groves et al. (1993) Nature 362:453-455). Further, epidermal stem cells have been used for grafting skin in burn patients (Green (1980) Scientific American).
Thus, differentiated stem cells with a desired potency and lineage specificity provides an unlimited supply of source material for tissue regeneration and repair and the treatment of a broad range of diseases.
To obtain specific cell lineages differentiated from the pluripotent stem cell, in vivo mechanisms to direct the differentiation into specific cell lineages have been used. For example, stem cells of a neuronal lineage have been isolated after modifying pluripotent stem cells with a reporter construct and then reintroducing them into an early stage embryo (Ott et at. (1994) J. Cell. Biochem. Supplement 18A:187). The reporter construct is expressed during neurogenesis and cells expressing the reporter gene are dissected out and placed in culture. Through in vivo mechanisms, this method allows for the isolation of cells committed to the neuronal lineage but, again, the dissected cells once placed in culture proceed to terminal differentiation.
U.S. Pat. No. 5,639,618 teaches a method of isolating a lineage-specific stem cell in vitro, by transfecting a pluripotent embryonic stem cell with a construct containing a regulatory region of a lineage-specific gene operably linked to a DNA encoding a reporter protein; culturing the pluripotent embryonic stem cell under conditions such that the pluripotent embryonic stem cell differentiates into a lineage-specific stem cell; and separating the cells which express the reporter protein from the other cells in the culture, wherein the cell which expresses the reporter protein is an isolated lineage-specific stem cell.
The most common system for stem cell identification involves the use of proteins expressed on the surface of cells as markers to identify cell types. Using fluorescently-tagged antibodies that bind to these surface proteins, cells expressing the appropriate proteins can be separated using fluorescent activated cell sorting (FACS) analysis. For example, Trempus, et al. ((April 2003) J. Invest. Dermatol. 120(4):501-11) teach the isolation of live CD34+ keratinocytes using antibodies to CD34 and alpha 6 integrin in combination with fluorescent-activated cell sorting. However, the identification and purification of stem cells using this type of method can be variable and difficult due to a lack of knowledge regarding the correlation between surface marker expression and stem cell specificity and further due to variations in antibody binding efficiencies (Alison et al. (2002) J. Pathol. 197:419-423). Although many characteristics of hematopoietic stem cells have been identified, the properties of most stem cells remain poorly defined, precluding the ability to identify markers common to all stem cells. Similarly, common markers distinguishing multipotent and pluripotent stem cells have not been heretofore defined (Jackson et al. (2002) J. Cell. Biochem. Suppl. 38:1-6). Thus, there is a need in the art for methods of identifying and isolating slow-cycling cells such as stem cells. The present invention addresses this long-felt need.